PP 1 - MICROSEQUENCING AND PEPTIDE MAPPING

A partial amino acid sequence can provide information about a proteins
used in the design of recombinant DNA probes. It is possible to obtain partial sequence data on as little as 10-100 pmoles of protein, which corresponds to 0.5-5 mg of a 50 kDa protein.

The N-terminus of proteins can be sequenced following separation by gel electrophoresis.This obviates the need to purify the protein to near homogeneity and allows one to take advantage of the high resolution of SDS gel electrophoresis. The requirements are sufficient protein which does not co-migrate with contaminating proteins .Proteins are electrophoretically transferred from the gels to a membrane support. The protein of interest is detected by staining and the band excised. Following extensive washing in water, the protein is sequenced directly from the membrane.

In some cases the N-termini of proteins are 'blocked' or internal sequence information is wanted. In such cases, proteins can be cleaved at specific residues through the use of proteases or chemical means. For example, CNBr cleaves proteins on the C-terminal side of cysteine residues. Many protease recognized specific amino acid residues and reproducibly cleaveproteins at specific sites. The resulting polypeptides are then isolated , absorbed onto glass fiber filters as a solid support, and the N-termini sequenced.

Peptide mapping. It is also possible to use site specific proteases to compare proteins and determine if they are related. The general procedure is to digest a protein with a protease and characterize the pattern of peptides formed. Each protein will exhibit a unique pattern of peptides. Such procedures are known as 'protein fingerprinting' or 'peptide mapping'. The peptides can be analyzed by HPLC, thin-layer chromatography or gel electrophoresis.

In two-dimensional peptide mapping the peptides are separated by TLC using
voltage electrophoresis followed by separation by high-voltage electrophoresis in a second dimension. Another form of peptide mapping involves excising a protein band from the gel and loading it on a second gel of higher acrylamide concentration. A protease is added to the well and the protein of interest is partially digested during the re-electrophoresis.

Proteins will exhibit a distinct pattern of smaller polypeptides. This method, often referred to as the Cleveland method, is rapid and easy to carry out and allows proteins to be directly compared in neighboring lanes. The pattern of polypeptides can be used to assess whether two proteins are similar or different.